Mechanisms for SU5416 as a radiosensitizer of endothelial cells

  • Authors:
    • Eun Ho Kim
    • Mi-Sook Kim
    • Youn Kyoung Jeong
    • Ilsung Cho
    • Seung Hoon You
    • Sung Ho Cho
    • Hanna Lee
    • Won-Gyun Jung
    • Hag Dong Kim
    • Joon Kim
  • View Affiliations

  • Published online on: August 21, 2015     https://doi.org/10.3892/ijo.2015.3127
  • Pages: 1440-1450
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Abstract

Endothelial cells (ECs), that comprise the tumor vasculature, are critical targets for anticancer radiotherapy. The aim of this work was to study the mechanism by which SU5416, a known anti-angiogenesis inhibitor, modifies the radiation responses of human vascular ECs. Two human endothelial cell lines (HUVEC and 2H11) were treated with SU5416 alone, radiation alone, or a combination of both. In vitro tests were performed using colony forming assays, FACS analysis, western blotting, immunohistochemistry, migration assay, invasion assays and endothelial tube formation assays. The combination of radiation and SU5416 significantly inhibited cell survival, the repair of radiation-induced DNA damage, and induced apoptosis. It also caused cell cycle arrest, inhibited cell migration and invasion, and suppressed angiogenesis. In this study, our results first provide a scientific rationale to combine SU5416 with radiotherapy to target ECs and suggest its clinical application in combination cancer treatment with radiotherapy.

Introduction

The cell-killing action of radiotherapy is purposed not only at tumor cells but also at endothelial cells (ECs) of the tumor vasculature that provides solid tumors with blood (1,2). The tumor vessel system, and in turn ECs, constitute a sensitive and critical target for tumor radiotherapy, resulting from the induction of radiation damage to the vasculature.

Targeting angiogenesis is an attractive therapeutic strategy to inhibit tumor growth, particularly since this approach has less probability of resulting in the development of drug resistance. Radiation sensitization of tumors has the ability to increase local tumor control and disease-free survival (3). The combination of radiotherapy and targeted drugs with anti-angiogenic or anti-vascular effects has been verified as an important aim for improving the therapeutic efficiency in cancer treatment (4). Recently, several compounds were investigated in preclinical studies and have since started clinical cancer trials that combine anti-angiogenic agents with ionizing radiation to improve the anticancer effect of radiation (59). One of such candidates, SU5416, is a selective inhibitor of the tyrosine kinase activity of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR and is currently in phase III clinical trials for the treatment of advanced malignancies by decreasing vascularization and growth of various human cancers (10,11). Single agent phase II clinical trials of SU5416 in patients with metastatic melanoma resulted in potential inhibitory effects on tumor vascularity (12), and an earlier study reported that the treatment reduced tumor growth and vascularization in an animal model of neuroblastoma (13). VEGF is known to be upregulated in various human tumors (1). The concept of combining anti-vascular compounds or angiogenesis inhibitors has also been widely studied for the combination of VEGF signaling inhibitors concurrently or sequentially together with radiotherapy (1422).

In this study, we investigated the mechanism by which SU5416 increased radiation-induced antitumor and anti-angiogenic effects by using endothelial cells as a representative model of the tumor vasculature. Along with the observed inhibition of VEGF signaling, we found that combination of SU5416 with radiation therapy markedly enhanced the therapeutic efficacy in endothelial cells.

Materials and methods

Antibodies and chemicals

Anti-cyclin B, anti-cyclin A, anti-cyclin E, anti-extracellular signal-regulated kinase (ERK), anti-Akt, anti-c-Jun N-terminal kinase (JNK), anti-p38 and anti-β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-cleaved PARP1 antibody, anti-cleaved caspase-3, anti-phospho-ERK, anti-phospho-Akt, anti-phospho-p38 and anti-phospho-JNK were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-γ-H2AX antibody was from Millipore. The angiogenesis inhibitor SU5416 was synthesized at Sugen Inc., as described previously (23). For in vitro experiments, it was dissolved in DMSO to make a 10 mmol/l stock solution and stored at −20°C.

Cell culture

Human umbilical vein endothelial cells (HUVECs) were maintained in endothelial cell basal medium (EGM-2) containing EGM-2 SingleQuot growth supplements (both from Cambrex) and maintained for no more than eight culture passages. The murine endothelial cell line 2H11 was maintained in DMEM plus 10% fetal bovine serum (FBS) in a humidified 10% CO2 environment.

Irradiation

Cells were plated in 60-mm dishes and incubated at 37°C under humidified conditions and 5% CO2 to 70–80% confluence. Cells were irradiated with a 137Cs gamma-ray source (Atomic Energy of Canada, Ltd., Ontario, ON, Canada) at a dose rate of 3.81 Gy/min.

Colony-forming assay

SU5416 (1 μmol/l) was preincubated for 6 h before radiation exposure and then incubated for a total of 72 h. After 14–20 days, colonies were stained with 0.4% crystal violet (Sigma, St. Louis, MO, USA). The plating efficiency (PE) represents the percentage of seeded cells that grew into colonies under the specific culture conditions of the given cell line. The survival fraction, expressed as a function of irradiation, was calculated as follows: survival fraction, colonies counted/(cells seeded × PE/100). The plating efficiency of HUVEC and 2H11 were 0.72±0.18 and 0.79±0.15. To evaluate the radio-sensitizing effects of SU5416, the ratio of radiation alone to radiation plus SU5416 was calculated as the dose (Gy) for radiation alone divided by the dose for radiation plus SU5416 at a surviving fraction of 10%.

Detection of apoptotic cells by Annexin V staining

After SU5416 preincubation, radiation was added to the cells, and the cells were subsequently incubated for 48 h. Cells were then washed with ice-cold PBS, trypsinized, and resuspended in 1X binding buffer [10 mm HEPES/NaOH (pH 7.4), 140 mm NaCl, and 2.5 mm CaCl2] at 1×106 cells/ml. Aliquots (100 μl) of cell solution were mixed with 5 μl Annexin V FITC (BD Pharmingen) and 10 μl propidium iodide (PI) stock solution (50 μg/ml in PBS) by gentle vortexing, followed by 15 min incubation at room temperature in the dark. Buffer (400 μl, 1X) was added to each sample and analyzed on a FACScan flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). A minimum of 10,000 cells was counted for each sample, and data analysis was performed in CellQuest software (BD Biosciences).

Immunohistochemistry

Immunohistochemistry was performed to determine the nuclear distribution of γ-H2AX in individual cells. Cells were grown on chamber slides for one day prior to irradiation or SU5416 treatments. After SU5416 exposure, cells were irradiated and treated for various time-points. All treatments were performed while cells remained attached to the slides, followed by fixing with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100 in PBS. Detection was performed after blocking the slides in 10% FBS/1% bovine serum albumin (BSA) for 1 h with a 1:1,000 dilution of fluorescein isothiocyanate (FITC)-labeled mouse monoclonal antibody against γ-H2AX, in the background-reducing antibody diluent (DAKO plus S3022) (both from Millipore, Billerica, MA, USA).

Western blotting

After SU5416 exposure, endothelial cells were irradiated and cultured for 1 and 24 h. Protein from treated cells was extracted with RIPA buffer, separated by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to nitrocellulose membranes. Membranes were blocked with 1% (v/v) nonfat dry milk in Tris-buffered saline with 0.05% Tween-20 and incubated with the indicated antibodies. Blots were reacted with primary antibodies at 1:1,000 dilutions and secondary antibodies at 1:5,000 dilutions. Immunoreactive protein bands were visualized by Enhanced Chemiluminescence (Amersham Biosciences) and scanned.

Wound healing scratch assay

Human endothelial cells were seeded onto 6-well plates (Corning) at 2.5×104 cells/well with 3 ml of medium. At two days, the monolayers were mechanically disrupted with a sterile 200 μl pipette tip. The assay was performed in duplicate and wells were photographed every 48 h prior to staining with 0.2% crystal violet. Cell migration was monitored using a Nikon Eclipse Ti microscope with a DS-Fi1 camera. The cells were counted using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

Invasion assay

The invasive ability in vitro was measured by using Transwell chambers, according to the manufacturer's protocol. Briefly, cells were seeded onto the membrane of the upper chamber of the Transwell at a concentration of 4×105/ml in 150 μl of medium and were left untreated or treated with the indicated doses of SU5416, radiation, or combine treatment for 24 h. The medium in the upper chamber was serum-free, whereas the lower chamber medium contained 10% FBS as a source of chemo-attractants. Cells that passed through the Matrigel-coated membrane were stained with Cell Stain Solution containing crystal violet supplied in the Transwell invasion assay (Chemicon, Millipore, GA, USA) and photographed after a 24-h incubation period.

Matrigel in vitro endothelial tube formation assay

Endothelial cell tube formation was carried out on Matrigel-coated chamber slides as described (24). The results of each assay were photographed (Nikon Eclipse Ti microscope with DS-Fi1 camera) at ×40 magnification. Tube formation was quantified by counting the number of connected cells in randomly selected fields at ×400 magnification with a microscope, and dividing that number by the total number of cells in the same field. Tube formation was quantified by counting the number of connected cells in randomly selected fields at ×400 magnification with a microscope, and dividing that number by the total number of cells in the same field.

Statistical analysis

All data were plotted as the mean ± standard error of the mean (SEM). Results of colony forming assays were analyzed using paired t-test with SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). All other data were analyzed by parametric repeated measure one-way ANOVA followed by Tukey's-HSD test (SPSS 18.0). Statistical significance was set at p<0.05.

Results

SU5416 radiosensitized ECs in vitro

To examine the effects of SU5416 on human endothelial cell radiosensitivity, we selected two endothelial cell lines, HUVEC and 2H11. We treated the EC cells with 1 μmol/l of SU5416 for 24 h prior to receiving different doses of irradiation. Using the concentration of SU5416 that showed a 20% decrease in cell survival (1 μM) after a 6 h pretreatment (data not shown), we further investigated the effects of SU5416 pretreatment on radiation-induced cell death. The results of colony forming assay of γ-irradiated EC cells with and without SU5416 pretreatment in form of a survival curves are shown in Fig. 1. The experimental survival fraction (S/S0) data points for γ-irradiated EC cells were fitted with linear quadratic dose (D) dependent relation given by S/S0=exp−(αD+βD2) where α and β are constants. The fitted values of α and β for γ-irradiated EC cells are given in Table I. The values from the fitted curves show that there is a significant decrease in survival fraction on combined treatment of SU5416 and radiation in comparison to those cells exposed to radiation only for 90% cell killing as shown in Table II.

Table I

Fitting parameters α and β for survival assay data.

Table I

Fitting parameters α and β for survival assay data.

Cell typeRadiation type α(Gy−1) β(Gy−1)
HUVECgamma-ray0.169±0.0400.039±0.012
gamma-ray + SU54160.463±0.0210.070±0.009
2H11gamma-ray0.166±0.6100.054±0.117
gamma-ray + SU54160.626±0.6100.025±0.124

[i] HUVEC, human umbilical vein endothelial cells.

Table II

Radiation dose required for 90% cell killing with and without SU5416.

Table II

Radiation dose required for 90% cell killing with and without SU5416.

Cell typeRadiation typeDose(Gy) for 90% cell killing without SU5416Dose(Gy) for 90% cell killing with SU5416
HUVECgamma-ray5.843.33
2H11gamma-ray5.163.25

[i] Radiosensitivity enhancement factor, REF corresponding to 90% cell killing is calculated to quantify the radiosensitization due to SU5416 using Eq: REF, radiation dose for 90% killing/radiation dose in presence of SU5416 for 90% killing. HUVEC, human umbilical vein endothelial cells.

The REF values for γ-irradiation of SU5416 treated EC cells are shown in Table III. These data showed that SU5416 had radiosensitizing effects on ECs in vitro.

Table III

REF and dose reduction values.

Table III

REF and dose reduction values.

Cell typeRadiation typeREF valueDose reduction (%)
HUVECgamma-ray1.7543
2H11gamma-ray1.5938

[i] HUVEC, human umbilical vein endothelial cells.

Effect of SU5416 on radiation-induced apoptosis

Ionizing radiation induces cell death after DNA damage (25). To investigate the induction of apoptosis after the combination treatment, we assessed early apoptosis by Annexin V and PI staining. Notably, 48 h of SU5416 and radiation exposure significantly increased the percentage of early apoptotic cells in endothelial cell lines (Fig. 2). We also examined whether SU5416 enhanced radiation cytotoxicity resulted from the further activation of the chief executioner of cell death, caspase-3 and PARP fragmentation, in endothelial cells (Fig. 2C). Our results showed that caspase-3 activation and PARP cleavage on treatment with SU5416 in combination with radiation was enhanced in comparison to that observed in the groups treated with SU5416 alone. We also monitored the expression of anti-apoptotic Bcl-2 protein and cell survival protein NF-κB and found that combination treatment clearly decreased the expression of Bcl-2 and NF-κB protein in both endothelial cell types (Fig. 2C).

Effects of SU5416 and radiation on cell cycle phase distribution

To investigate what cellular mechanisms may underlie the enhanced ionizing radiation-induced cell death following the combined treatment of SU5416, we examined the cell cycle profiles (Fig. 3A and B). Results showed that combination treatments caused alterations in distribution of cells in different phases of cell cycle in both endothelial cell lines. SubG1 phase, indicating the apoptotic population, was only moderately changed in endothelial cells following treatment with SU5416; however, the combined treatment caused accumulation of cells in subG1 phase in endothelial cells. Combination therapy caused the most accumulation of cells at G2/M phases, suggesting the highest increase of cell cycle arrest at G2/M phase in both endothelial cell lines. We also studied the expression of cell cycle regulator following combined treatment with SU5416 and radiation (Fig. 3C). Western blotting showed that radiation alone showed an accumulation of cyclin B1 involved in the G2/M transition, whereas SU5416 alone reduced the expression of the regulator. In contrast, cyclin B1 expression was attenuated by 24 h of SU5416 treatment, regardless of the radiation dose (Fig. 3C).

Influence of SU5416 on radiation-induced DNA damage and DNA repair activity

We further evaluated the DNA damage response by analyzing the expression of the damage-responsive protein H2AX. Histone H2AX is phosphorylated at Ser139 (γ-H2AX) in response to double-strand break (DSB) processing. As expected, SU5416-pretreated cells showed a higher level of ionizing radiation-induced γ-H2AX (Fig. 4), since it is known that SU5416 increases ionizing radiation-induced DSB. The expression of γ-H2AX was consistently observed until 24 h after radiation exposure in the presence of SU5416. Both endothelial cells after the combined treatment with SU5416 and radiation showed damaged DNA foci, which appeared at 1 h after radiation exposure and remained even until 24 h (Fig. 4A). Furthermore, SU5416 treatment itself did not alter the induction or subsequent disappearance of foci at any time point examined.

Effects of SU5416 and radiation on mitogen-activated protein kinase (MAPK) expression in ECs

Members of the MAPK, ERK and JNK are well known to be involved in DNA damage responses (26,27). We performed western blotting to assess the levels of the phosphorylated form of pro-survival marker ERK and the pro-apoptotic marker JNK (Fig. 5). The level of p-ERK in IR irradiated cells was significantly increased, whereas, sharply decreased in the combination treatment cells.

Since the radioprotective response of ECs includes Akt activation (28,29), we theorized that the enhanced irradiation-induced apoptosis observed in SU5416-treated ECs was caused by the inhibition of Akt activation. In line with this, an increase of the active phosphorylated form of Akt protein (p-Akt) was found in EC cells subjected to IR or SU5416 treatment; however, combination treatment reduced the level of the induced p-Akt (Fig. 5). This inhibition of p-Akt may be responsible for the radiosensitizing effects of SU5416 by enhancing the susceptibility of G2/M arrested cells to undergo apoptosis in response to the IR induced DNA damage.

Effect of SU5416 and radiation on cell motility and cell invasion

We next evaluated the effects of SU5416 on the invasive and migratory capacities of endothelial cells using a scratch assay. Notably, compared with treatment with SU5416 or radiation alone, combination treatment significantly inhibited cell migration (Fig. 6A). We also performed Matrigel invasion assay to examine the effect of SU5416 combination treatment on tumor cell invasiveness and found that combination treatment was highly effective at inhibiting tumor cell invasion in both endothelial lines (Fig. 6B).

Combination treatment significantly inhibits angiogenesis

The production of tubular structures is another critical process in angiogenesis; therefore, we investigated the effects of SU5416 on HUVEC and 2H11 tube formation. SU5416 treatment not only quantitatively reduced the number of formed tubes in EC cultures, but also altered the morphology of the cells (Fig. 7). These effects were significantly greater in combination-treated cells as compared to each treatment alone, suggesting at least an additive inhibition of tube formation (Fig. 7).

Discussion

Combinations of radiotherapy with drugs are being investigated to enhance the cure rates and decrease side effects. In this regard, drugs with anti-angiogenic effects are of great interest. The importance of angiogenesis in the progression of human tumors has been presented by many outstanding academic studies describing the relationship between angiogenic tumor phenotypes and patient survival (30). These studies suggested that the number of microvessels in primary tumors possibly predict the prognosis of various tumors such as lung (31,32), breast (33,34), bladder (35) and colon (36) carcinomas, and in tumors of the oral cavity (37). Thus, control of angiogenesis has been identified as an critical approach for the therapeutic applications of human cancers (38,39). The vascular endothelium is very resistant to the effects of radiation, so VEGF may cause enhanced resistance to radiation-induced damage (40). Therefore, preclinical studies propose that anti-angiogenic agents increase tumor control in response to radiation (6). Many such compounds are already in clinical studies or have been approved for the treatment of certain malignancies (41). However, it is still unclear which combinations of signaling inhibitors would be the most effective as an anticancer regimen and which of these would benefit from a combination with radiotherapy. In this context, we showed in ECs that the combination of VEGF signaling inhibition increases the anticancer effects of each monotherapy. We also found that radiotherapy in combination with VEGF signaling inhibition highly increases anti-angiogenic and antitumor effects of the respective drug therapies. In these studies, we used SU5416 as an inhibitor of VEGF signaling.

Recently, clinical studies have shown that the combination of SU5416 and radiation may offer clinical gains in patients with human cancer cell types (14); however, the mechanism underlying this effect appears to be somewhat more complex than that predicted in previous studies. Here, we provide a scientific rationale for the clinical application of SU5416 as a radiosensitizer in ECs. We have studied the various mechanisms by which SU5416 may increase the therapeutic efficacy of radiation by inhibiting tumor cell survival, cell cycle regulation, DNA repair activity, tumor cell invasiveness and angiogenesis in ECs.

Previous studies show that SU5416 combined with radiation significantly decrease the clonogenic survival abilities and enhance the radiosensitivity of ECs by activating apoptosis through PARP and caspase-3 (Figs. 1 and 2). When SU5416 was treated after radiation, ECs failed to undergo mitosis seemingly due to a block in transition from G2 to M phase that may result from the downregulation of cyclin B1 (Fig. 3). The irradiation of cells leads to electron scattering through the Compton effect, which subsequently causes DNA breaks (42). H2AX phosphorylation, a marker of DNA DSBs, was estimated as an indication of the radiation-induced DNA damage response (4345). The results show that the combined treatment delayed the clearance of γ-H2AX, suggesting that SU5416 maintains DNA damage, thus increasing radiosensitivity (Fig. 4).

Migration of cancer cells is also a major event in meta-static cascade of cancers. Cell migration is a highly integrated process that is important for the growth of cancer cells in various organs of the body; therefore, we examined the effect of our combination therapy in inhibiting the migratory capacity of ECs. VEGF-2 expression plays an important role in cell migration by initiating many intracellular signaling pathways (46). Our data showed that decreases in VEGF-2 expression are associated with a decrease in the migratory potential of ECs following exposure to combination of SU5416 and radiation (Fig. 6). In ECs, ionizing radiation leads to phosphatidylinositol 3-kinase (PI3K)/Akt pathway activation that contributes to post-irradiation cell survival (28,29). Some tumor-derived growth factors, particularly VEGF and basic fibroblast growth factor (bFGF), can enhance the radioresistance of ECs (47), but this can also occur through PI3K/Akt signaling (48). Inhibitors of PI3K, wortmannin, and LY294002, are well known to enhance the radiosensitivity of ECs (28); therefore, appropriate pharmacologic blockers of the PI3K/Akt pathway could be applied to radiosensitize the tumor vasculature. Akt is a pro-survival gene required for the G2/M transition (49,50), and oncogenic Akt can enhance the survival of cells after DNA damage by overcoming this checkpoint block, as well as the apoptosis induced by DNA damage (51). Recent evidence shows that many radiosensitizers possess anticancer effects through the inhibition of Akt (52), and VEGF is known as an important target of SU5416. VEGF dominantly acts on angiogenesis via the PI3K/Akt and MAPK signaling cascade (53). We observed that the VEGF2 phosphorylation raised by irradiation was downregulated by both combinations of SU5416 and radiation (Fig. 5). ERK is a key downstream component of the RAF/MEK/ERK signaling pathway and aberrant signaling through the ERK pathway was able to increase cell immortalization, proliferation and resistance to radiation (54). Western blot analysis demonstrated that radiation led to ERK activation, which was suppressed by the post-irradiation treatment of SU5416. Active form of Akt has been studied to indirectly inhibit JNK, a kinase known to control apoptosis (55). Consistent with previous studies, we observe that IR- and combination-treated EC cells show a negative correlation between p-Akt and p-JNK protein levels. ERK pathway activation is rescued with radioresistance (56), and the observable decrease in ERK activation after combination treatment likely sensitizes the cells to apoptosis. Our data are consistent with a model whereby combination treatment sensitizes the cells by relieving IR-induced resistance to apoptosis in ECs by first limiting the Akt survival pathway, and subsequently inducing JNK activation, thus committing the cells to an apoptotic fate. Moreover, VEGF treatment of endothelial cells significantly enhanced the tube formation on growth factor reduced Matrigel (Fig. 7). Our data demonstrate that SU5416 in combination treatment also inhibited VEGF-mediated endothelial cells tube formation (in vitro angiogenesis assay).

In conclusion, our investigation demonstrated that the anti-angiogenic compound SU5416 had an effective therapeutic radiosensitizing potential in endothelial cells. This radiosensitizing effect was associated with an inhibition of cell survival, cell cycle regulation, DNA repair activity, tumor cell invasiveness, and angiogenesis. We have provided evidence for the molecular basis of chemoradiation treatment; however, in vivo mouse model experiments should be carried out to minimize the possible complications in clinical applications. Furthermore, as the radiosensitizing effect of SU5416 in photon beam treatment is well known, it will be necessary to compare its sensitizing effect to proton or carbon beams in particle radiation to enhance the biological efficiency and safety of these forms of radiotherapy.

Acknowledgements

This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP; no.NRF-2014R1A1A3053958,NRF2015R1A2A1A05001823).

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October-2015
Volume 47 Issue 4

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Spandidos Publications style
Kim EH, Kim M, Jeong YK, Cho I, You SH, Cho SH, Lee H, Jung W, Kim HD, Kim J, Kim J, et al: Mechanisms for SU5416 as a radiosensitizer of endothelial cells. Int J Oncol 47: 1440-1450, 2015
APA
Kim, E.H., Kim, M., Jeong, Y.K., Cho, I., You, S.H., Cho, S.H. ... Kim, J. (2015). Mechanisms for SU5416 as a radiosensitizer of endothelial cells. International Journal of Oncology, 47, 1440-1450. https://doi.org/10.3892/ijo.2015.3127
MLA
Kim, E. H., Kim, M., Jeong, Y. K., Cho, I., You, S. H., Cho, S. H., Lee, H., Jung, W., Kim, H. D., Kim, J."Mechanisms for SU5416 as a radiosensitizer of endothelial cells". International Journal of Oncology 47.4 (2015): 1440-1450.
Chicago
Kim, E. H., Kim, M., Jeong, Y. K., Cho, I., You, S. H., Cho, S. H., Lee, H., Jung, W., Kim, H. D., Kim, J."Mechanisms for SU5416 as a radiosensitizer of endothelial cells". International Journal of Oncology 47, no. 4 (2015): 1440-1450. https://doi.org/10.3892/ijo.2015.3127